Project Summary Specialized sensory organs contain functionally dedicated cell types that detect relevant stimuli and relay information to the nervous system. In this proposal, we ask if this concept also pertains to the gut epithelium, which constitutes one of the largest exposed surface areas of the human body and is in contact with a diverse chemical environment. Indeed, numerous chemical changes in the gut lumen have been associated with visceral pain, including irritants, endogenous inflammatory molecules, and microbiota-produced metabolites. Despite growing interest in the gut-neural axis, relatively little is known about molecular mechanisms underlying chemosensory transduction by the gut epithelium, or how this information is transmitted to the nervous system. Serotonergic enterochromaffin (EC) cells are rare, but highly specialized entities within the gut epithelium that have been implicated in visceral pain but have eluded detailed characterization due, in part, to their paucity. To circumvent these limitations, we generated intestinal organoids from a transgenic mouse in which EC cells are marked with a fluorophore, enabling us to carry out detailed single-cell profiling of these cells in the context of a native tissue environment. Our preliminary data show that these cells are electrically excitable, polymodal chemosensory detectors of the gut that engage in direct synaptic interactions with sensory nerve fibers to transduce information about intestinal state. In these proposed studies, we will define intrinsic EC cell electrophysiological properties, chemosensory transduction mechanisms, and serotonin release mechanisms (Aim 1) and utilize this information to investigate the physiological effects of EC activation on and associated neural pathways (Aim 2). Finally, we will obtain genetic access to EC cells and use chemogenetic tools to examine their contribution to visceral pain (Aim 3). This work will elucidate EC cell chemosensory mechanisms and examine their role in visceral pain to provide a mechanistic foundation for understanding how the gut epithelium communicates with the nervous system. This molecular foundation is critical for uncovering basic mechanisms that contribute to pathophysiology underlying visceral pain disorders, such as irritable bowel syndrome. Proposed experimental approaches for this award combine my expertise in cellular physiology and biophysics with new training in genetics and GI physiology, allowing me to address significant biological questions and identify novel molecular mechanisms. A unique mentorship team with extensive experience in signal transduction, pain, synaptic physiology, and GI physiology will provide expert guidance and an ideal environment for proposed scientific and professional development. Thus, the training supported by this award will be critical to establishing a unique and important independent research program in neuroscience and gastrointestinal physiology.